Kinetics of formation and stability of {Pt(dien)}2+ complexes with octamer and 14-mer DNA oligonucleotides containing a GG sequence

 Reaction of [Pt(dien)Cl]⁺ (1) with the 14-mer oligonucleotide 5′-d(ATACATGGTACATA) (I) gave rise to two major species which corresponded to the 5′-G and 3′-G platinated monofunctional adducts, and a minor amount of the bis-platinated adduct formed during the later stages of the reaction. The reaction of (1) with the related octamer 5′-d(ATACATGG) (II) was also investigated. Kinetic data obtained by HPLC showed that the 5′-G and 3′-G bases of the 14-mer oligonucleotide were platinated at similar rates: the second-order rate constant is 53×10^–2 M^–1 s^–1 at 298 K in 0.1 M NaClO₄. However, the platination rate of 5′-G of the octamer (II) (k=69×10^–2 M^–1 s^–1) was enhanced by a factor of three compared to the rate of platination at 3′-G (k=22×10^–2 M^–1 s^–1). All the adducts were separated by HPLC and characterized by NMR spectroscopy, enzymatic digestion and MALDI-TOF mass spectrometry. ¹H and ¹⁵N NMR shifts suggest that there are distinct conformational differences between 14-mer duplexes platinated at 5′-G (I5′ _ds) and 3′–G (I3′ _ds). Molecular mechanics modelling indicates that rotation around the Pt-N7 bond is more restricted in the case of the 5′-G adduct than in that of the 3′-G adduct. The binding of {Pt(dien)}²⁺ to 5′-GN7 and 3′-GN7 in the monofunctional adducts of (I) was shown to be reversible upon the addition of high concentrations of chloride ions. Received: 3 July 1998 / Accepted: 10 November 1998     

Mechanism of dithionite reduction of the R2 protein of E. coli and mouse ribonucleotide reductase: evidence for reduction of FeIII 2 prior to the tyrosyl radical

 Dithionite has been found to reduce directly (without mediators) the Escherichia coli R2 subunit of ribonucleotide reductase. With dithionite (∼10 mM) in large excess, the reaction at 25  °C is complete in ∼10 h. Preparations of E. coli R2 have an Fe^III ₂ (met-R2) component in this work at ∼40% levels, alongside the fully active enzyme Fe^III ₂ . . . Tyr*, which has a tyrosyl radical at Tyr-122. In the pH range studied (7–8) the kinetics are biphasic. Rate laws for both phases give [S₂O₄ ^2–] and not [S₂O₄ ^2–]^1/2 dependencies, and saturation kinetics are observed for the first time in R2 studies. No dependence on pH was detected. The kinetics (25  °C) of the first phase are reproduced in separate experiments using only met-R2, with association of S₂O₄ ^2– to met-R2, K=330 M^–1, occurring prior to electron transfer, k _et=4.8×10^–4 s^–1, I=0.100 M (NaCl). The second phase assigned to the reaction of Fe^III ₂ . . . Tyr* with S₂O₄ ^2– gives K=800 M^–1 and k _et=5.6×10^–5 s^–1. Bearing in mind the substantially smaller reduction potential for Fe^III ₂ compared to Tyr*, this is a quite remarkable finding, with implications similar to those already reported for the reaction of R2 with hydrazine, but with additional information provided by the saturation kinetics. The similarity in rates for the two phases (∼fourfold difference) suggests that reduction of Fe^III ₂ is occurring in both cases, and since S₂O₄ ^2– is involved a two-equivalent change is proposed with the formation of Fe^II ₂ . . . Tyr* in the case of active R2. As a sequel to the second phase, intramolecular reduction of the strongly oxidising Tyr* by the Fe^II ₂ is rapid, and further decay of Fe^IIFe^III is also fast. There is no stable mouse met-R2 form, and the single-phase reaction with dithionite gives saturation kinetics with K=208 M^–1 and k _et=1.7±10^–3 s^–1. Mechanistic implications, including the applicability of a pathway for electron transfer via Fe_A, are considered. Received: 25 February 1998 / Received: 20 August 1998     

Direct assessment of water exchange on a Gd(III) chelate bound to a protein

 The Gd(III) complex of 4-pentylbicyclo[2.2.2]octane-1-carboxyl-di-l-aspartyl-lysine-derived DTPA, [GdL(H₂O)]^2–, binds to serum albumin in vivo, through hydrophobic interaction. A variable temperature ¹⁷O NMR, EPR, and Nuclear Magnetic Relaxation Dispersion (NMRD) study resulted in a water exchange rate of k ²⁹⁸ _ex=4.2×10⁶ s^–1, and let us conclude that the GdL complex is identical to [Gd(DTPA)(H₂O)]^2– in respect to water exchange and electronic relaxation. The effect of albumin binding on the water exchange rate has been directly evaluated by ¹⁷O NMR. Contrary to expectations, the water exchange rate on GdL does not decrease considerably when bound to bovine serum albumin (BSA); the lowest limit can be given as k _{ex, GdL-BSA}=k _ex, GdL / 2. In the knowledge of the water exchange rate for the BSA-bound GdL complex, the analysis of its NMRD profile at 35  °C yielded a rotational correlation time of 1.0 ns, one order of magnitude shorter than that of the whole protein. This value is supported by the longitudinal ¹⁷O relaxation rates. This indicates a remarkable internal flexibility, probably due to the relatively large distance between the protein- and metal-binding moieties of the ligand. Received: 25 June 1998 / Accepted: 11 August 1998     

Effects of trans-pyridine ligands on the interactions of RuII and RuIII ammine complexes N7-coordinated to purine nucleosides and DNA

 DNA binding by trans-[(H₂O)(Pyr)(NH₃)₄Ru^II]²⁺ (Pyr=py, 3-phpy, 4-phpy, 3-bnpy, 4-bnpy) is highly selective for G⁷ with K _G=1.1×10⁴ to 2.8×10⁴, with the more hydrophobic Pyr ligands exhibiting slightly higher binding. A strong dependence on ionic strength indicates that ion-pairing with DNA occurs prior to binding. At μ=0.05, d[Ru^II-DNA]/dt=k[Ru^II][DNA], where k=0.17–0.21 M^–1s^–1 with the various Pyr ligands. The air oxidation of [(py)(NH₃)₄Ru^II] _n -DNA to [(py)(NH₃)₄Ru^III] _n -DNA at pH 6 occurs with a pseudo-first-order rate constant of k _obs=5.6×10^–4s^–1 at μ=0.1, T=25  °C. Strand cleavage of plasmid DNA appears to occur by both Fenton/Haber-Weiss chemistry and by base-catalyzed routes, some of which are independent of oxygen. Base-catalyzed cleavage is more efficient than O₂ activation at neutral pH and involves the disproportionation of covalently bound Ru^III and, in the presence of O₂, Ru-facilitated autoxidation to 8-oxoguanine. Disproportionation of [py(NH₃)₄Ru^III] _n -DNA occurs according to the rate law: d[Ru^II–G_DNA]/dt=k ₀[Ru^III–G_DNA]+k ₁[Ru^III–G_DNA][OH^–], where k ₀=5.4×10^–4s^–1 and k ₁=8.8 M^–1s^–1 at 25  °C, μ=0.1. The appearance of [(Gua)(py)(NH₃)₄Ru^III] under argon, which occurs according to the rate law: d[Ru^III–G]/dt=k ₀[Ru^III–G_DNA]+k ₁[OH^–][Ru^III–G_DNA] (k ₀=5.74×10^–5 s^–1, k ₁=1.93×10^–2M^–1s^–1 at T=25  °C, μ=0.1), is consistent with lysis of the N-glycosidic bond by Ru^IV-induced general acid hydrolysis. In air, the ratio of [Ru-8-OG]/[Ru-G] and their net rates of appearance are 1.7 at pH 11, 25  °C. Small amounts of phosphate glycolate indicate a minor oxidative pathway involving C4′ of the sugar. In air, a dynamic steady-state system arises in which reduction of Ru^IV produces additional Ru^II. Received: 11 November 1998 / Accepted: 3 March 1999     

Kinetics and mechanism for reduction of anticancer-active tetrachloroam(m)ine platinum(IV) compounds by glutathione

trans -[PtCl₄(NH₃)(thiazole)] (1), trans-[PtCl₄(cha)(NH₃)] (2), cis-[PtCl₄(cha)(NH₃)] (3) (cha =cyclohexylamine), and cis-[PtCl₄(NH₃)₂] (4) has been investigatedat 25 °C in a 1.0 M aqueous medium at pH 2.0–5.0 (1) and 4.5–6.8 (2–4) using stopped-flow spectrophotometry. The redox reactions follow the second-order rate law , where k is a pH-dependent rate constant and [GSH]_tot the total concentration of glutathione. The reduction takes place via parallel reactions between the platinum(IV) complexes and the various protolytic species of glutathione. The pH dependence of the redox kinetics is ascribed to displacement of these protolytic equilibria. The thiolate species GS⁻ is the major reductant under the reaction conditions used. The second-order rate constants for reduction of compounds 1–4 by GS⁻ are (1.43±0.01)×10⁷, (3.86±0.03)×10⁶, (1.83±0.01)×10⁶, and (1.18±0.01)×10⁶M⁻¹s⁻¹, respectively. Rate constants for reduction of 1 by the protonated species GSH are more than five orders of magnitude smaller. The mechanism for the reductive elimination reactions of the Pt(IV) compounds is proposed to involve an attack by glutathione on one of the mutually trans coordinated chloride ligands, leading to two-electron transfer via a chloride-bridged activated complex. The kinetics results together with literature data indicate that platinum(IV) complexes with a trans Cl-Pt-Cl axis are reduced rapidly by glutathione as well as by ascorbate. In agreement with this observation, cytotoxicity profiles for such complexes are very similar to those for the corresponding platinum(II) product complexes. The rapid reduction within 1 s of the platinum(IV) compounds with a trans Cl-Pt-Cl axis to their platinum(II) analogs does not seem to support the strategy of using kinetic inertness as a parameter to increase anticancer activity, at least for this class of compounds. Received: 8 December 1999 / Accepted: 15 February 2000     

Kinetic studies on the reactions of Fusarium galactose oxidase with five different substrates in the presence of dioxygen

 Reactions (25  °C) of galactose oxidase, GOase_ox from Fusarium NRRL 2903 with five different primary-alcohol-containing substrates RCH₂OH:- D-galactose (I) and 2-deoxy-d-galactose (II) (monosaccharides); methyl-β-d-galactopyranoside (III) (glycoside);d-raffinose (IV) (trisaccharide); and dihydroxyacetone (V) have been studied in the presence of O₂. The GOase_ox state has a tyrosyl radical coordinated at a square-pyramidal Cu^II active site, and is a two-equivalent oxidant. Reactant concentrations were [GOase_ox] (0.8–10 μM), RCH₂OH (1.0–6.0 mM), and O₂ (0.14–0.29 mM), with I=0.100 M (NaCl). The reactions, monitored at 450 nm by stopped-flow spectrophotometry, terminated with depletion of the O₂. Each trace was fitted to the competing reactions GOase_ox+RCH₂ OH → GOase_redH₂+RCHO (k ₁), and GOase_redH₂+O₂→ GOase_ox+H₂O₂ (k ₂), with GOase_redH₂ written as the doubly protonated two-electron-reduced Cu^I product. It was necessary to avoid auto-redox interconversion of GOase_ox and GOase_semi . Information obtained at pH 7.5 indicates a 5 : 95 (ox : semi) "native" mix equilibration complete in ∼3 h. At pH >7.5, rate constants 10^–4 k ₁ / M^–1 s^–1 for the reactions of GOase_ox with (I) (1.19), (II) (1.07), (III) (1.29), (IV) (1.81), (V) (2.94) were determined. On decreasing the pH to 5.5, k ₁ values decreased by factors of up to a half, and acid dissociation pK _as in the range 6.6–6.9 were obtained. UV-Vis spectrophotometric studies on GOase_ox gave an independently determined pK _a of 6.7. No corresponding reactions of the Tyr495Phe variant were observed, and there are no similar UV-Vis absorbance changes for this variant. The pK _a is therefore assigned to protonation of Tyr-495 which is a ligand to the Cu. The rate constant k ₂ (1.01×10⁷ M^–1 s^–1) is independent of pH in the range 5.5–9.0 investigated, suggesting that H⁺ (or H-atoms) for the O₂ → H₂O₂ change are provided by the active site of GOase_red . The Cu^I of GOase_red is less extensively complexed, and a coordination number of three is likely. Received: 4 February 1997 / Accepted: 16 May 1997     

Characterization of the cadmium complex of peptide 49–61: a putative nucleation center for cadmium-induced folding in rabbit liver metallothionein IIA

 The synthetic peptide fragment containing residues 49–61 of rabbit liver metallothionein II (MT-II) (Ac-Ile-Cys-Lys-Gly-Ala-Ser-Asp-Lys-Cys-Ser-Cys-Cys-Ala-COOH), which includes the only sequential four cysteines bound to the same metal ion in Cd₇MT, forms a stable, monomeric Cd-peptide complex with 1 : 1 stoichiometry (Cd:peptide) via Cd-thiolate interactions. This represents the first synthesis of a single metal-binding site of MT independent of the domains. The ¹¹¹Cd NMR chemical shift at 716 ppm indicates that the ¹¹¹Cd²⁺ in the metal site is terminally coordinated to four side-chain thiolates of the cysteine residues. The pH of half dissociation for this Cd-peptide derivative, ∼3.3, demonstrates an affinity similar to that for Cd₇MT. Molecular mechanics calculations show that the thermodynamically most stable folding for this isolated Cd²⁺ center has the same counterclockwise chirality (Λ or S) observed in the native holo-protein. These properties are consistent with its proposed role as a nucleation center for cadmium-induced protein folding. However, the kinetic reactivity of the CdS₄ structure toward 5,5′-dithiobis(5-nitrobenzoate) (DTNB) and EDTA is greatly increased compared to the complete cluster (α-domain or holo-protein). The rate law for the reaction with DTNB is rate=(k _uf +k _1,f +k _2,f [DTNB])[peptide], where k _uf=0.15 s^–1, k _1,f=2.59×10^–3s^–1, and k _2,f=0.88 M^–1s^–1. The ultrafast step (uf), observable only by stopped-flow measurement, is unprecedented for mammalian (M₇MT) and crustacean (M₆MT) holo-proteins or the isolated domains. The accommodation of other metal ions by the peptide indicates a rich coordination chemistry, including stoichiometries of M-peptide for Hg²⁺, Cd²⁺, and Zn²⁺, M₂-peptide for Hg²⁺ and Au⁺, and (Et₃PAu)₂-peptide. Received: 9 December 1998 / Accepted: 20 May 1999     

The Relative Rates of Thiol–Thioester Exchange and Hydrolysis for Alkyl and Aryl Thioalkanoates in Water

This article reports rate constants for thiol–thioester exchange (k _ex), and for acid-mediated (k _a), base-mediated (k _b), and pH-independent (k _w) hydrolysis of S-methyl thioacetate and S-phenyl 5-dimethylamino-5-oxo-thiopentanoate—model alkyl and aryl thioalkanoates, respectively—in water. Reactions such as thiol–thioester exchange or aminolysis could have generated molecular complexity on early Earth, but for thioesters to have played important roles in the origin of life, constructive reactions would have needed to compete effectively with hydrolysis under prebiotic conditions. Knowledge of the kinetics of competition between exchange and hydrolysis is also useful in the optimization of systems where exchange is used in applications such as self-assembly or reversible binding. For the alkyl thioester S-methyl thioacetate, which has been synthesized in simulated prebiotic hydrothermal vents, k _a = 1.5 × 10⁻⁵ M⁻¹ s⁻¹, k _b = 1.6 × 10⁻¹ M⁻¹ s⁻¹, and k _w = 3.6 × 10⁻⁸ s⁻¹. At pH 7 and 23°C, the half-life for hydrolysis is 155 days. The second-order rate constant for thiol–thioester exchange between S-methyl thioacetate and 2-sulfonatoethanethiolate is k _ex = 1.7 M⁻¹ s⁻¹. At pH 7 and 23°C, with [R″S(H)] = 1 mM, the half-life of the exchange reaction is 38 h. These results confirm that conditions (pH, temperature, pK _a of the thiol) exist where prebiotically relevant thioesters can survive hydrolysis in water for long periods of time and rates of thiol–thioester exchange exceed those of hydrolysis by several orders of magnitude.     

Type I blue copper proteins as enantioselective quenchers of the photoluminescence of Δ,Λ-Eu(pyridine-2,6-dicarboxylate)3 3–: azurin from Pseudomonas aeruginosa and its Met44→Lys mutant, amicyanin from Paracoccus versutus and parsley plastocyanin

 The dynamic quenching of the luminescence of racemic Eu(III)(pyridine-2,6-dicarboxylate=dpa)₃ ^3– by the title proteins is investigated and the enantioselectivity of the proteins in the quenching of the Δ and Λ enantiomers of Eu(dpa)₃ ^3– is determined. The two diastereomeric quenching rate constants pertaining to azurin (k _q ^Δ=3.3×10⁶, k _q ^Λ=2.7×10⁶ M^–1s^–1, pH 7.2, ionic strength I=22 mM) are lower than for its Met→44Lys mutant (k _q ^Δ=1.9×10⁷, k _q ^Λ=1.4×10⁷ M^–1 s^–1, same pH and I), indicating that energy transfer occurs from Eu(dpa)₃ ^3– to the Cu(II) centre when the luminophore is bound to the hydrophobic patch of the protein near residue 44. The enantioselectivity remains unaltered by the mutation: k _q ^Δ/k _q ^Λ=1.27±0.04, so Lys44 is probably not in direct contact with the Eu chelate. The I and pH dependence of k _q indicate that the lysine residue interacts electrostatically with Eu(dpa)₃ ^3–. For plastocyanin the quenching rates are of the order of 10⁶ M^–1 s^–1; for amicyanin they are two orders of magnitude larger (k _q ^Δ=12×10⁷, k _q ^Λ=11×10⁷ M^–1 s^–1, pH 7.2, I=22 mM). The variation of k _q is attributed to differences in the charge distribution on the proteins, which influences the binding of the luminophore to the protein surface. For amicyanin the anion binding site near Lys59 and Lys60 may be involved in the energy transfer. Received: 16 June 1998 / Accepted: 18 September 1998     

Kinetic studies of electron transfer in the cyt b 5 : metHb system

 The kinetics of methemoglobin reduction by cytochrome b ₅ has been studied by stopped-flow and saturation transfer NMR. A forward rate constant k _f = 2.44×10⁴ M^–1 s^–1 and a reverse rate constant k _b = 540 M^–1s^–1 have been observed at 10 mm, pH 6.20, 25  °C. The ratio k _f/k _b = k _eq = 43.6 is in good agreement with the equilibrium constant calculated from the electrochemical potential between cyt b ₅ and methemoglobin. A bimolecular collisional mechanism is proposed for the electron transfer from cyt b ₅ to methemoglobin based on the kinetic data analysis. The dependence of the rate constants on ionic strengths supports such collisional mechanism. It is also found that the reaction rate strongly depends on the conformations of methemoglobin. Received: 20 February 1996 / Accepted: 4 June 1996     

Stereoselective and driving-force-dependent photoinduced electron-transfer reactions of zinc myoglobin with optically active N,N′-dimethylcinchoninium and N,N′-dimethylcinchonidinium ions

In order to understand the detailed mechanism of the stereoselective photoinduced electron-transfer (ET) reactions of zinc-substituted myoglobin (ZnMb) with optically active molecules by flash photolysis, we designed and prepared new optically active agents, such as N,N′-dimethylcinchoninium diiodide ([MCN]I₂) and N,N′-dimethylcinchonidinium diiodide ([MCD]I₂). The photoexcited triplet state of ZnMb, ³(ZnMb)*, was successfully quenched by [MCN]²⁺ and [MCD]²⁺ ions to form the radical pair of ZnMb cation (ZnMb^·+) and reduced [MCN]^·+ and [MCD]^·+, followed by a thermal back ET reaction to the ground state. The rate constants (k _q) for the ET quenching at 25 °C were obtained as k _q(MCN)=(1.9±0.1)×10⁶ M⁻¹ s⁻¹ and k _q(MCD)=(3.0±0.2)×10⁶ M⁻¹ s⁻¹, respectively. The ratio of k _q(MCD)/k _q(MCN)=1.6 indicates that the [MCD]²⁺ preferentially quenches ³(ZnMb)*. The second-order rate constants (k _b) for the thermal back ET reaction from [MCN]^·+ and [MCD]^·+ to ZnMb^·+ at 25 °C were k _b(MCN)=(0.79±0.04)×10⁸ M⁻¹ s⁻¹ and k _b(MCD)=(1.0±0.1)×10⁸ M⁻¹ s⁻¹, respectively, and the selectivity was k _q(MCD)/k _q(MCN)=1.3. Both quenching and thermal back ET reactions are controlled by the ET step. In the quenching reaction, the energy differences of ΔΔH ^≠(MCD–MCN) and ΔΔS ^≠(MCD–MCN) at 25 °C were obtained as −1.1 and 0 kJ mol⁻¹, respectively. On the other hand, ΔΔH ^≠(MCD–MCN)=11±2 kJ mol⁻¹ and TΔΔS ^≠(MCD–MCN)=−10±2 kJ mol⁻¹ were given in the thermal back ET reaction. The highest stereoselectivity of 1.7 for [MCD]^·+ found at low temperature (10 °C) was due to the ΔΔS ^≠ value obtained in the thermal back ET reaction. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Steady-state kinetics, micellar effects, and the mechanism of peroxidase-catalyzed oxidation of n -alkylferrocenes by hydrogen peroxide

 Kinetics of the steady-state oxidation of n–alkylferrocenes (alkyl = H, Me, Et, Bu and C₅H₁₁) by H₂O₂ to form the corresponding ferricenium cations catalyzed by horseradish peroxidase has been studied in micellar systems of Triton X-100, CTAB, and SDS, mostly at pH 6.0 and 25  °C. The rate of oxidation of ferrocenes with longer alkyl radicals is too slow to be measured. The reaction obeying the [RFc]:[H₂O₂] = 2 : 1 stoichiometry is strictly first-order in both HRP and RFc in a wide concentration range. The corresponding observed second-order rate constants k, which refer to the interaction of the peroxidase compound II (HRP-II) with RFc, decrease with the elongation of the alkyl substituent R, and this in turn is accompanied by an increase in the formal redox potentials E°′ in the same medium. Increasing the surfactant concentration lowers the rate constants k, the effect being due to the nonproductive binding of RFc to micelles rather than to enzyme inactivation. The micellar effects are accounted for in terms of the Berezin pseudo-phase model of micellar catalysis applied to the interaction of enzyme with organometallic substrates. The oxidation was found to occur primarily in the aqueous pseudo-phase and the calculated intrinsic second-order rate constants k _w are (1.9 ± 0.5)×10⁵, (2.7 ± 0.1)×10⁴, and (5.9 ± 0.6)×10³ M^–1 s^–1 for HFc, EtFc, and n–BuFc, respectively. The data obtained were used for estimating the self-exchange rate constants for the HRP-II/HRP couple in terms of the Marcus formalism. Received: 15 July 1996 / Accepted: 15 November 1996     

Magnesium complexes of the antiviral 9-[2-(phosphonomethoxy)ethyl]adenine (PMEA) and of its 1-, 3-, and 7-deaza analogues in aqueous solution

 The stability constants of the 1 : 1 complexes formed between Mg²⁺ and the anions of the N1, N3, and N7 deaza derivatives of 9-[2-(phosphonomethoxy)ethyl]adenine (PA^2–), i.e., of Mg(H;PA)⁺ and Mg(PA), were determined by potentiometric pH titration in aqueous solution (25  °C; I=0.1 M, NaNO₃) and compared with previous results [Sigel H, et al. (1992) Helv Chim Acta 75 : 2634–2656], obtained under the same conditions, for the corresponding complexes of 9-[2-(phosphonomethoxy)ethyl]adenine (PMEA^2–) and (phosphonomethoxy)ethane (PME^2–). Based on the analysis of a microconstant scheme it is concluded that in the monoprotonated complexes, Mg(H;PA)⁺, Mg²⁺ is coordinated to a significant part at the nucleobase, H⁺ being at the phosphonate group. By making use of log K ^Mg _Mg(R-PO3) versus pK ^H _H(R-PO3) straight-line plots (also obtained previously; see above) for simple phosphonates and phosphate monoesters, it is shown that all the Mg(PA) complexes, including those with PMEA^2– and PME^2–, are more stable than expected on the basis of the basicity of the ―PO^2– ₃ group. This proves that, to some extent, five-membered chelates, Mg(PA)_cl/O, involving the ether oxygen of the ―CH₂―O―CH₂―PO^2– ₃ chain are formed; their formation degree amounts to about 30–40% in equilibrium with the isomer having only a phosphonate-Mg²⁺ coordination. In the case of Mg(1-deaza-PMEA), probably a further isomer occurs in which also N3 of the nucleobase participates. The different properties between the Mg(PA) species and the Mg(AMP) complex are discussed. Received: 26 January 1998 / Accepted: 19 May 1998     

Rates of intercalator-driven platination of DNA determined by a restriction enzyme cleavage inhibition assay

A restriction enzyme cleavage inhibition assay was designed to determine the rates of DNA platination by four non-cross-linking platinum–acridine agents represented by the formula [Pt(am₂)LCl](NO₃)₂, where am is a diamine nonleaving group and L is an acridine derived from the intercalator 1-[2-(acridin-9-ylamino)ethyl]-1,3-dimethylthiourea (ACRAMTU). The formation of monofunctional adducts in the target sequence 5′-CGA was studied in a 40-base-pair probe containing the EcoRI restriction site GAATTC. The time dependence of endonuclease inhibition was quantitatively analyzed by polyacrylamide gel electrophoresis. The formation of monoadducts is approximately 3 times faster with double-stranded DNA than with simple nucleic acid fragments. Compound 1 (am₂ is ethane-1,2-diamine, L is ACRAMTU) reacts with a first-order rate constant of k _obs = 1.4 ± 0.37 × 10⁻⁴ s⁻¹ (t _1/2 = 83 ± 22 min). Replacement of the thiourea group in ACRAMTU with an amidine group (compound 2) accelerates the rate by fourfold (k _obs = 5.7 ± 0.58 × 10⁻⁴ s⁻¹, t _1/2 = 21 ± 2 min), and introduction of a propane-1,3-diamine nonleaving group results in a 1.5-fold enhancement in reactivity (compound 3, k _obs = 2.1 ± 0.40 × 10⁻⁴ s⁻¹, t _1/2 = 55 ± 10 min) compared with the prototype. Derivative 4, containing a 4,9-disubstituted acridine threading intercalator, was the least reactive compound in the series (k _obs = 1.1 ± 0.40 × 10⁻⁴ s⁻¹, t _1/2 = 104 ± 38 min). The data suggest a correlation may exist between the binding rates and the biological activity of the compounds. Potential pharmacological advantages of rapid formation of cytotoxic monofunctional adducts over the common purine–purine cross-links are discussed.     

Recombinant horseradish peroxidase isoenzyme C: the effect of distal haem cavity mutations (His42→Leu and Arg38→Leu) on compound I formation and substrate binding

 Horseradish peroxidase isoenzyme C (HRPC) mutants were constructed in order to understand the role of two key distal haem cavity residues, histidine 42 and arginine 38, in the formation of compound I and in substrate binding. The role of these residues as general acid-base catalysts, originally proposed for cytochrome c peroxidase by Poulos and Kraut in 1980 was assessed for HRPC. Replacement of histidine 42 by leucine [(H42L)HRPC*] decreased the apparent bimolecular rate constant for the reaction with hydrogen peroxide by five orders of magnitude (k ₁ = 1.4×10² M^–1s^–1) compared with both native-glycosylated and recombinant forms of HRPC (k ₁ = 1.7×10⁷ M^–1s^–1). The first-order rate constant for the heterolytic cleavage of the oxygen-oxygen bond to form compound I was estimated to be four orders of magnitude slower for this variant. Replacement of arginine 38 by leucine [(R38L)HRPC*] decreased the observed pseudo-first-order rate constant for the reaction with hydrogen peroxide by three orders of magnitude (k ₁ = 1.1×10⁴ M^–1s^–1), while the observed rate constant of oxygen bond scission was decreased sixfold (k ₂ = 142 s^–1). These rate constants are consistent with arginine 38 having two roles in catalysing compound I formation: firstly, promotion of proton transfer to the imidazole group of histidine 42 to facilitate peroxide anion binding to the haem, and secondly, stabilisation of the transition state for the heterolytic cleavage of the oxygen-oxygen bond. These roles for arginine 38 explain, in part, why dioxygen-binding globins, which do not have an arginine in the distal cavity, are poor peroxidases. Binding studies of benzhydroxamic acid to (H42L)HRPC* and (R38L)HRPC* indicate that both histidine 42 and arginine 38 are involved in the modulation of substrate affinity. Received: 21 July 1995 / Accepted: 27 November 1995     

The reaction mechanism of nitrosothiols with copper(I)

Copper and other transition metal ions and their complexes are catalysts for the decomposition of nitrosothiols. In this way they catalyze the biological functions of nitrosothiols. The kinetics and mechanism of the reaction of two nitrosothiols, S-nitrosothiolactic acid and S-nitrosoglutathione (GSNO), with copper(I) are reported. The kinetics of the reaction of Cu(MeCN) _n ⁺ (n=0–3) with the nitrosothiols were studied. The results indicate that Cu⁺ _aq is the active species in the GSNO system, with k(Cu⁺ _aq+GSNO)=(9.4 ±2.0)×10⁷ dm³ mol⁻¹ s⁻¹. The results also indicate that the Cu(MeCN) _n ⁺ (n=0–3) complexes react with S-nitrosothiolactic acid. Transient species are formed in these processes. The results suggest that these species contain copper(I) and thiol. The results shed light on the catalytic role of copper complexes in the decomposition of S-nitrosothiols. Received 10 April 1999 / Accepted 17 December 1999     

Isolation, culture and regeneration of avocado (Persea americana Mill.) protoplasts

Protoplasts were isolated from embryogenic suspension cultures derived from avocado (Persea americana Mill.) zygotic embryos and nucellus in an enzyme digestion solution consisting of 1% cellulase Onozuka RS, 1% Macerase R10, 0.2% Pectolyase Y-23, 0.7 M mannitol. 24.5 mM CaCl₂, 0.92 mM NaH₂PO₄ and 6.25 2-[N-morpholino]ethanesulfonic acid (1.5 ml) mixed with 0.7 M MS^–8P (2.5 ml). MS^-8P medium consisted of Murashige and Skoog salts without NH₄NO₃, 1 mg l^–1 thiamine HCl, 100 mg l^–1 myo-inositol, 3.1 g l^–1 glutamine and 8P organic addenda. Medium osmolarity was adjusted with 0.15 M sucrose and 0–0.55 M mannitol. Protoplast yields of 3.5×10⁶ protoplasts g^–1 were obtained. Growth and development of the protoplasts were significantly affected by osmolarity, nitrogen source, plating density and culture medium dilution. Under optimum conditions, proembryos developed directly from embryogenic protoplasts and subsequently into somatic embryos. Optimum conditions for somatic embryo development included the culture of protoplasts at a density of 0.8–1.6×10⁵ ml^–1 in 0.4 M MS^–8P for 2–3 weeks, followed by subculture in 0.15 M MS^–8P at a diluted density of 20–40× for 1 month in darkness to obtain somatic embryos. Mature somatic embryos were recovered on semisolid medium; however, a low frequency of plantlet recovery (≤1%) from protoplast-derived somatic embryos was observed. Received: 9 February 1998 / Revision received: 4 May 1998 / Accepted: 15 May 1998     

Trans-pyridine tetraammine complexes of RuII and RuIII with N7-coordninated purine nucleosides

 The synthesis, spectroscopic, and electrochemical properties of trans-[L(Pyr)(NH₃)₄Ru^II/III] (Pyr=py, 3-phpy, 4-phpy, 3-bnpy, or 4-bnpy; L=H₂O, Guo, dGuo, 1MeGuo, Gua, Ino, or G⁷-DNA) are reported. As expected, the Pyr ligand slows DNA binding by trans-[(H₂O)(Pyr)(NH₃)₄Ru^II]²⁺ relative to [(H₂O)(NH₃)₅Ru^II]²⁺ and favors reduction of Ru^III by about 150 mV. The pyridine ligand also promotes the disproportionation of Ru^III to afford the corresponding complexes of Ru^II and, presumably, Ru^IV. For L=Ino, disproportionation follows the rate law: d[Ru^II]/dt=k ₀[Ru^III]+k ₁[OH^–][Ru^III], k ₀=(2.7±0.7)×10^–4s^–1 and k ₁=70±1 M^–1s^–1. Received: 11 May 1998 / Accepted: 3 March 1999     

Kinetics and thermodynamics of peroxidase- and laccase-catalyzed oxidation of N-substituted phenothiazines and phenoxazines

N -substituted phenothiazines (PTs) and phenoxazines (POs) catalyzed by fungal Coprinus cinereus peroxidase and Polyporus pinsitus laccase were investigated at pH 4–10. In the case of peroxidase, an apparent bimolecular rate constant (expressed as k _cat/K _m) varied from 1 ×10⁷M⁻¹s⁻¹to 2.6×10⁸M⁻¹s⁻¹ at pH 7.0. The constants for PO oxidation were higher in comparison to PT. pH dependence revealed two or three ionizable groups with pK _a values of 4.9–5.7 and 7.7–9.7 that significantly affected the activity of peroxidase. Single-turnover experiments showed that the limiting step of PT oxidation was reduction of compound II and second-order rate constants were obtained which were consistent with the constants at steady-state conditions. Laccase-catalyzed PT and PO oxidation rates were lower; apparent bimolecular rate constants varied from 1.8×10⁵M⁻¹s⁻¹ to 2.0×10⁷M⁻¹s⁻¹ at pH 5.3. PO constants were higher in comparison to PT, as was the case with peroxidase. The dependence of the apparent bimolecular constants of compound II or copper type 1 reduction, in the case of peroxidase or laccase, respectively, was analyzed in the framework of the Marcus outer-sphere electron-transfer theory. Peroxidase-catalyzed reactions with PT, as well as PO, fitted the same hyperbolic dependence with a maximal oxidation rate of 1.6×10⁸M⁻¹s⁻¹ and a reorganization energy of 0.30 eV. The respective parameters for laccase were 5.0×10⁷M⁻¹s⁻¹ and 0.29 eV. Received: 20 September 1999 / Accepted: 24 February 2000     

Kinetics of N-substituted phenothiazines and N-substituted phenoxazines oxidation catalyzed by fungal laccases

Laccase-catalyzed oxidation of N-substituted phenothiazines and N-substituted phenoxazines was investigated at pH 5.5 and 25°C. The recombinant laccase from Polyporus pinsitus (rPpL) and the laccase from Myceliophthora thermophila (rMtL) were used. The dependence of initial reaction rate on substrate concentration was analyzed by applying the laccase action scheme in which the laccase native intermediate (NI) reacts with a substrate forming reduced enzyme. The reduced laccase produces peroxide intermediate (PI) which in turn decays to the NI. The calculated constant (k_ox) values of the PI formation are (6.1±3.1)×10⁵ M⁻¹s⁻¹ for rPpL and (2.5±0.9)×10⁴ M⁻¹s⁻¹ for rMtL. The bimolecular constants of the reaction of the native intermediate with electron donor (kred) vary in the interval from 2.2×10⁵ to 2.1×10⁷ M⁻¹s⁻¹ for rPpL and from 1.3×10² to 1.8×10⁵ M^-1s⁻¹ for rMtL. The larger reactivity of rPpL in comparison to rMtL is associated with the higher redox potential of type I Cu of rPpL. The variation of k_red values for both laccases correlates with the change of the redox potential of substrates. Following outer sphere (Marcus) electron transfer mechanism the calculated activationless electron transfer rate and the apparent reorganization energy are 5.0×107 M⁻¹s⁻¹ and 0.29 eV, respectively.